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This study aims to uncover the molecular mechanisms regulating ginkgolic acid biosynthesis in Ginkgo biloba episperm.

March 2, 2026Horticulture Research0 citationsOpen Access

Single-nucleus sequencing and spatial metabolomics analysis reveal the regulatory mechanism of ginkgolic acid biosynthesis in the episperm of Ginkgo Biloba

ZFZhi FengCentral South University of Forestry and Technology ZYZhi YaoCentral South University of Forestry and Technology BWBin WangHunan Normal University

Key Points

This study aims to uncover the molecular mechanisms regulating ginkgolic acid biosynthesis in Ginkgo biloba episperm.
Performed mass spectrometry imaging of Ginkgo biloba seed
Constructed a single-cell expression atlas of the episperm
Identified seven cell types in the episperm
Conducted pseudotime analysis and gene expression trajectory analysis
Developed a molecular interaction network related to ginkgolic acid biosynthesis
Ginkgolic acids mainly accumulate in secretory cavities of the episperm
Identified GbWRKY35 as a core transcription factor in the biosynthesis pathway
Confirmed GbWRKY35's regulatory role through interaction with signaling proteins
Revealed that GbWRKY35 stimulates GbAAE16 expression for ginkgolic acid production

Abstract

Abstract Ginkgo biloba is a singular and relict gymnosperm indigenous to China. Its distinctive fleshy episperm is rich in unique metabolites, ginkgolic acids, which protect the developing seed from biotic stresses. The unique nature of the tissue and its metabolites has made it highly challenging to elucidate the molecular and cellular mechanisms governing ginkgolic acid biosynthesis and regulation. In this study, we performed the mass spectrometry imaging of G. biloba seed, revealing that ginkgolic acids primarily accumulate in the secretory cavities of the episperm. We constructed a single-cell expression atlas of the G. biloba episperm, and identifying seven cellular types: meristem cells, subepidermal cells, lignified cells, trancheid cells, parenchymal cells, secretory cavity cells, and epidermis cells. Based on the analysis of upregulated gene expression in secretory cavity cells, pseudotime analysis of cell differentiation and gene expression trajectory analysis, we precisely identified the key enzyme-encoding genes highly associated with ginkgolic acid biosynthesis. This approach elucidated the cellular and molecular mechanisms underlying secretory cell differentiation, secretory cavity formation, and ginkgolic acid biosynthesis and accumulation in response to exogenous jasmonic acid induction. By constructing a molecular interaction network, it was determined that the GbWRKY35, encoded by Gb₂5334, is the core transcription factor. We further identified the signaling proteins that interact with GbWRKY35, confirming its central positive regulatory role in ginkgolic acid biosynthesis. As a core transcription factor, GbWRKY35 regulates ginkgolic acid biosynthesis through stimulating the expression of GbAAE16. This study provides the first spatially resolved investigation into the molecular and cellular regulatory mechanisms of ginkgolic acid biosynthesis in the episperm under jasmonic acid induction.

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